莱茵衣藻胞外聚合物的提取和红外光谱表征
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摘要
胞外聚合物(EPS)是微生物细胞代谢过程中产生的高分子物质,其中的多种官能团通过络合、转化等反应改变周围环境的元素形态和活性,在污染生物修复中具有应用前景.以模式微藻——莱茵衣藻为研究材料,采用加热法、NaOH法、EDTA法、阳离子交换树脂(CER)法、高速离心法提取莱茵衣藻的EPS,比较分析每种方法得到的EPS的含量及其组成成分,并结合LIVE/DEAD BacLight染色和激光共聚焦显微镜观察细胞活性,以确定莱茵衣藻EPS最适宜的提取方法.结果表明,不同提取方法得到的EPS各组分含量有显著差异,5种方法的提取总量依次为NaOH法>加热法>CER法>EDTA法>离心法.荧光染色和激光共聚焦显微镜检测结果表明,相对于NaOH法,加热法对微藻细胞的破坏程度较小,且傅里叶红外光谱分析结果表明,加热法提取的EPS出现N-H、C=O或C-N(蛋白质)和C-H、C-O-C、RHC(OH)(OR)(糖类)等吸收峰.本研究表明加热法是莱茵衣藻EPS的最适宜提取方法,EPS的官能团信息sk可为后续研究莱茵衣藻EPS与重金属之间的相互作用奠定基础.
Extracellular polymeric substances are macromolecules produced in the metabolic processes of microbial cells.Many functional groups from extracellular polymeric substances(EPS) might change the speciation and activities of elements in the surrounding environment via complexation and transformation. These processes indicate that EPS have potential application in the bioremediation of pollution. In this study, we extracted the EPS of Chlamydomonas reinhardtii using heating, NaOH, EDTA, cation exchange resin(CER), and high-speed centrifugation methods. To develop a suitable method for EPS extraction from C. reinhardtii, the contents and composition of the EPS obtained from each method were compared.The cell viabilities after EPS removal under different extractions were observed using LIVE/DEAD BacLight staining and laser confocal microscopy. The results showed that the contents of EPS components were significantly different among the extraction methods. The total amount of EPS extracted by different methods was ranked as follows: NaOH > heating > cation exchange resin(CER) > EDTA > centrifugation. LIVE/DEAD BacLight staining and laser confocal microscopy showed that the C. reinhardtii cells were only slightly deactivated by heating compared with the NaOH treatment. The Fourier-transform infrared spectroscopy(FTIR) analysis demonstrated that the EPS extracted by the heating method had functional groups, such as N-H, C=O, or C-N(protein) and C-H, C-O-C, RHC(OH)(OR)(carbohydrate). In summary, heating was the most suitable EPS extraction method for C. reinhardtii. The information of EPS functional groups provided a basis for further studies on the interactions between EPS and heavy metals in this microalga.
Extracellular polymeric substances are macromolecules produced in the metabolic processes of microbial cells.Many functional groups from extracellular polymeric substances(EPS) might change the speciation and activities of elements in the surrounding environment via complexation and transformation. These processes indicate that EPS have potential application in the bioremediation of pollution. In this study, we extracted the EPS of Chlamydomonas reinhardtii using heating, NaOH, EDTA, cation exchange resin(CER), and high-speed centrifugation methods. To develop a suitable method for EPS extraction from C. reinhardtii, the contents and composition of the EPS obtained from each method were compared.The cell viabilities after EPS removal under different extractions were observed using LIVE/DEAD BacLight staining and laser confocal microscopy. The results showed that the contents of EPS components were significantly different among the extraction methods. The total amount of EPS extracted by different methods was ranked as follows: NaOH > heating > cation exchange resin(CER) > EDTA > centrifugation. LIVE/DEAD BacLight staining and laser confocal microscopy showed that the C. reinhardtii cells were only slightly deactivated by heating compared with the NaOH treatment. The Fourier-transform infrared spectroscopy(FTIR) analysis demonstrated that the EPS extracted by the heating method had functional groups, such as N-H, C=O, or C-N(protein) and C-H, C-O-C, RHC(OH)(OR)(carbohydrate). In summary, heating was the most suitable EPS extraction method for C. reinhardtii. The information of EPS functional groups provided a basis for further studies on the interactions between EPS and heavy metals in this microalga.
引文
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2 Zhou Y, Nguyen BT,Zhou C, Straka L,Lai YS,Xia S,Rittmann BE. The distribution of phosphorus and its transformations during batch growth of Synechocystis[J]. Water Res, 2017, 122:355-362
3 Sheng GP, Yu HQ, Li XY. Extracellular polymeric substances(EPS)of microbial aggregates in biological wastewater treatment systems:a review[J]. Biotechnol Adv, 2010, 28(6):882-894
4 Sendra M, Moreno-Garrido I, Yeste MP, Gatica JM, Blasco J. Toxicity of TiO2, in nanoparticle or bulk form to freshwater and marine microalgae under visible light and UV-A radiation[J]. Environ Pollut, 2017, 227:39-48
5 Yu Q, Fein JB. Sulfhydryl binding sites within bacterial extracellular polymeric substances[J]. Environ Sci Technol, 2016, 50(11):5498-5505
6梁君,宋文成,马金宝.微生物胞外聚合物对水中As(V)的吸附性能研究[J].环境工程,2016(s1):226-229[Liang J,Song WC,Ma JB. Study on sorption performances of As(V)by extracellular polymeric substances[J]. Environ Engin, 2016(s1):226-229]
7刘翠霞,胡智泉,郭雪自.Zn2+对藻类生物膜生长生理特性影响研究[J].生态环境学报,2013(5):838-843[Liu CX,Hu ZQ,Guo XB.Effects of Zn2+on the growth physiological characteristics of algal biofilm[J]. Ecol Environ Sci, 2013, 22(5):838-843]
8 Pan XL, Liu J, Song WJ, Zhang DY. Biosorption of Cu(Ⅱ)to extracellular polymeric substances(EPS)from Synechoeystis sp.:a fluorescence quenching study[J]. Front End Sci Eng, 2012, 6(4):493-497
9刘静,张道勇,潘响亮,王立英.藻菌生物膜胞外聚合物(EPS)与Al3+的配位作用机理[J].应用与环境生物学报,2009, 15(3):347-350[Liu J, Zhang DY, Pan XL, Wang LY. Characterization of the complexation between Al3+and extracellular polymeric substances prepared from algabacteria biofilm[J]. Chin JAppl Environ Biol, 2009,15(3):347-350]
10康福星,龙健,王倩.微生物胞外聚合物对水体重金属和富营养元素的环境生化效应研究展望[J].应用与环境生物学报,2010(1):129-134[Kang FX, Long J, Wang Q. Environmental and biochemical effects of microbial extracellular polymeric substances on the heavy metals and eutrophic elements in water areas:a review[J]. Chin J Appl Environ Biol, 2010(1):129-134]
11 Brown MJ, Lester JN. Comparison of bacterial extracellular polymerextraction methods[J]. Appl Environ Microbiol, 1980, 40(2):179-185
12 Wingender J, Neu TR, Flemming HC. Microbial Extracellular Polymeric Substances[M]. Berlin:Springer, 1999
13 Ni L,Li D,Rong S,Su L,Zhou W,Wang P,Wang C,Li S,Acharya K.Characterization of extracellular polymeric substance(EPS)fractions produced by Microcystis aeruginosa under the stress of linoleic acid sustained-release microspheres[J]. Environ Sci Pollut Res, 2017, 24(26):21091-21102
14 Kang F, Alvarez PJ, Zhu D. Microbial extracellular polymeric substances reduce Ag+to silver nanoparticles and antagonize bactericidal activity[J]. Environ Sci Technol, 2013, 48(1):316-322
15 Xu H,Cai H, Yu G,Jiang H. Insights into extracellular polymeric substances of cyanobacterium Microcystis aeruginosa using fractionation procedure and parallel factor analysis[J]. Water Res, 2013,47(6):2005-2014
16 Song W,Zhao C,Zhang D, Mu SY, Pan XL. Different resistance to UV-B radiation of extracellular polymeric substances of two cyanobacteria from contrasting habitats[J]. Front Microbiol, 2016, 7:1208-1216
17 Stuart RK,Mayali X,Lee JZ,Craig-Everroad R,Hwang M,Bebout BM, Weber PK, Pett-Ridge J, Thelen MP. Cyanobacterial reuse of extracellular organic carbon in microbial mats[J]. ISME J, 2016, 10(5):1240-1251
18 Xu H, Yu G, Jiang H. Investigation on extracellular polymeric substances from mucilaginous cyanobacterial blooms in eutrophic freshwater lakes[J]. Chemosphere, 2013, 93(1):75-81
19 Cao B, Ahmed B, Kennedy DW, Wang Z, Shi L, Marshall MJ,Fredrickson JK, Isern NG, Majors PD, Beyenal H. Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1biofilms to U(VI)immobilization[J]. Environ Sci Technol, 2011, 45(13):5483-5490
20 Jain R,Jordan N,Weiss S,Foerstendorf H,Heim K,Kacker R, Hiibner R, Kramer H, Van Hullebusch ED, Farges F,Lens PN. Extracellular polymeric substances govern the surface charge of biogenic elemental selenium nanoparticles[J]. Environ Sci Technol, 2015, 49(3):1713-1720
21王硕,于水利,徐巧,付强,李激.好氧颗粒污泥特性、应用及形成机理研究进展[J].应用与环境生物学报,2014, 20(4):732-742[Wang S, Yu SL, Xu Q, Fu Q, Li J. Characteristics, application and formation mechanisms of aerobic granular sludge:recent advances[J]. Chin J Appl Environ Biol, 2014, 20(4):732-742]
22 More TT, Yadav JSS, Yan S, Tyagi RD, Surampalli RY. Extracellular polymeric substances of bacteria and their potential environmental applications[J]. J Environ Manage, 2014, 144:1-25
23 Bafana A. Characterization and optimization of production of exopolysaccharide from Chlamydomonas reinhardtii[J]. Carbohydr.Polym, 2013, 95(2):746-752
24 Fernandez M, Sanchez J. Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451[J]. J Microbiol Meth, 2002, 49(2):207-208
25 Dai YF, Xiao Y, Zhang EH, Liu LD, Qiu L, You LX, DummiMahadevan G, Chen BL, Zhao F. Effective methods for extracting extracellular polymeric substances from Shewanella oneidensis MR-1[J]. Water Sci Technol, 2016, 74(12):2987-2996
26 Kang F, Qu X, Alvarez PJ, Zhu D. Extracellular saccharide-mediated reduction of Au3+to gold nanoparticles:new insights for heavy metals biomineralization on microbial surfaces[J]. Environ Sci Technol, 2017,51(5):2776-2785
27张宇,梁彦秋,贾春云,姜春阳,焦阳.不同方法提取PAHs高效降解菌EPS的特性[J].生态学杂志,2014, 33(4):1027-1033[Zhang Y, Lang YQ, Jia CY, Jiang CY, Jiao Y. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods[J]. Chin JEcol, 2014, 33(4):1027-1033]
28 Hou J, Yang Y, Wang P, Wang C, Lu M, Wang X, Lu B, You G, Liu Z.Effects of CeO2, CuO, and ZnO nanoparticles on physiological features of Microcystis aeruginosa and the production and composition of extracellular polymeric substances[J]. Environ Sci Pollut Res Int, 2017,24(1):226-235
29 Xu H, Jiang H, Yu G, Yang L. Towards understanding the role of extracellular polymeric substances in cyanobacterial Microcystis aggregation and mucilaginous bloom formation[J]. Chemosphere, 2014,117:815-822
30杨敏.微生物胞外聚合物的生成及其对树脂特性的影响研究[D].昆明:昆明理工大学,2012[Yang M. The generation of microbial extracellular polymeric substances and its influence on resin characteristic[D].Kunming:Kunming University of Science of Technology, 2012]
31 Liu L,Qin B,Zhang Y, Zhu G,Gao G, Huang Q, Yao X. Extraction and characterization of bound extracellular polymeric substances fromcultured pure cyanobacterium(Microcystis wesenbergii)[J]. J Environ Sci, 2014, 26(8):1725-1732
32 Wang LL,Wang LF,Ren XM,Ye XD,Li WW,Yuan SJ,Sun M,Sheng GP, Yu HQ, Wang XK. pH dependence of structure and surface properties of microbial EPS[J]. Environ Sci Technol, 2012, 46(2):737-744
33 Morgan JW, Forster CF, Evison L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges[J]. Water Res, 1990, 24(6):743-750
34 Sun M, Li WW, Mu ZX, Wang HL, Yu HQ, Li YY, Harada H. Selection of effective methods for extracting extracellular polymeric substances(EPSs)from Bacillus megaterium TF10[J]. Sep Purif Technol, 2012, 95:216-221
35郑昊.莱茵衣藻对纳米氧化铜在水环境中的分布规律及去除效能的影响[D].泰安:山东农业大学,2016[Zheng H. Effects of Chlamydomonas reinhardtii on removal and distribution of CuO nanoparticle[D]. Tai'an:Shandong Agricultural University, 2016]
36 Zhou K, Hu Y, Zhang L, Yang K, Lin D. The role of exopolymeric substances in the bioaccumulation and toxicity of Ag nanoparticles to algae[J]. Sci Rep, 2016, 6:32998-33009
37慕亚南.自然水体生物膜及其胞外聚合物对Cu2+的吸附研究[D].无锡:江南大学,2016[Mu YN. Adsorption of Cu2+by natural biofilms associated with extracellular polymeric substances(EPS)[D]. Wuxi:Jiangnan University,2016]
2 Zhou Y, Nguyen BT,Zhou C, Straka L,Lai YS,Xia S,Rittmann BE. The distribution of phosphorus and its transformations during batch growth of Synechocystis[J]. Water Res, 2017, 122:355-362
3 Sheng GP, Yu HQ, Li XY. Extracellular polymeric substances(EPS)of microbial aggregates in biological wastewater treatment systems:a review[J]. Biotechnol Adv, 2010, 28(6):882-894
4 Sendra M, Moreno-Garrido I, Yeste MP, Gatica JM, Blasco J. Toxicity of TiO2, in nanoparticle or bulk form to freshwater and marine microalgae under visible light and UV-A radiation[J]. Environ Pollut, 2017, 227:39-48
5 Yu Q, Fein JB. Sulfhydryl binding sites within bacterial extracellular polymeric substances[J]. Environ Sci Technol, 2016, 50(11):5498-5505
6梁君,宋文成,马金宝.微生物胞外聚合物对水中As(V)的吸附性能研究[J].环境工程,2016(s1):226-229[Liang J,Song WC,Ma JB. Study on sorption performances of As(V)by extracellular polymeric substances[J]. Environ Engin, 2016(s1):226-229]
7刘翠霞,胡智泉,郭雪自.Zn2+对藻类生物膜生长生理特性影响研究[J].生态环境学报,2013(5):838-843[Liu CX,Hu ZQ,Guo XB.Effects of Zn2+on the growth physiological characteristics of algal biofilm[J]. Ecol Environ Sci, 2013, 22(5):838-843]
8 Pan XL, Liu J, Song WJ, Zhang DY. Biosorption of Cu(Ⅱ)to extracellular polymeric substances(EPS)from Synechoeystis sp.:a fluorescence quenching study[J]. Front End Sci Eng, 2012, 6(4):493-497
9刘静,张道勇,潘响亮,王立英.藻菌生物膜胞外聚合物(EPS)与Al3+的配位作用机理[J].应用与环境生物学报,2009, 15(3):347-350[Liu J, Zhang DY, Pan XL, Wang LY. Characterization of the complexation between Al3+and extracellular polymeric substances prepared from algabacteria biofilm[J]. Chin JAppl Environ Biol, 2009,15(3):347-350]
10康福星,龙健,王倩.微生物胞外聚合物对水体重金属和富营养元素的环境生化效应研究展望[J].应用与环境生物学报,2010(1):129-134[Kang FX, Long J, Wang Q. Environmental and biochemical effects of microbial extracellular polymeric substances on the heavy metals and eutrophic elements in water areas:a review[J]. Chin J Appl Environ Biol, 2010(1):129-134]
11 Brown MJ, Lester JN. Comparison of bacterial extracellular polymerextraction methods[J]. Appl Environ Microbiol, 1980, 40(2):179-185
12 Wingender J, Neu TR, Flemming HC. Microbial Extracellular Polymeric Substances[M]. Berlin:Springer, 1999
13 Ni L,Li D,Rong S,Su L,Zhou W,Wang P,Wang C,Li S,Acharya K.Characterization of extracellular polymeric substance(EPS)fractions produced by Microcystis aeruginosa under the stress of linoleic acid sustained-release microspheres[J]. Environ Sci Pollut Res, 2017, 24(26):21091-21102
14 Kang F, Alvarez PJ, Zhu D. Microbial extracellular polymeric substances reduce Ag+to silver nanoparticles and antagonize bactericidal activity[J]. Environ Sci Technol, 2013, 48(1):316-322
15 Xu H,Cai H, Yu G,Jiang H. Insights into extracellular polymeric substances of cyanobacterium Microcystis aeruginosa using fractionation procedure and parallel factor analysis[J]. Water Res, 2013,47(6):2005-2014
16 Song W,Zhao C,Zhang D, Mu SY, Pan XL. Different resistance to UV-B radiation of extracellular polymeric substances of two cyanobacteria from contrasting habitats[J]. Front Microbiol, 2016, 7:1208-1216
17 Stuart RK,Mayali X,Lee JZ,Craig-Everroad R,Hwang M,Bebout BM, Weber PK, Pett-Ridge J, Thelen MP. Cyanobacterial reuse of extracellular organic carbon in microbial mats[J]. ISME J, 2016, 10(5):1240-1251
18 Xu H, Yu G, Jiang H. Investigation on extracellular polymeric substances from mucilaginous cyanobacterial blooms in eutrophic freshwater lakes[J]. Chemosphere, 2013, 93(1):75-81
19 Cao B, Ahmed B, Kennedy DW, Wang Z, Shi L, Marshall MJ,Fredrickson JK, Isern NG, Majors PD, Beyenal H. Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1biofilms to U(VI)immobilization[J]. Environ Sci Technol, 2011, 45(13):5483-5490
20 Jain R,Jordan N,Weiss S,Foerstendorf H,Heim K,Kacker R, Hiibner R, Kramer H, Van Hullebusch ED, Farges F,Lens PN. Extracellular polymeric substances govern the surface charge of biogenic elemental selenium nanoparticles[J]. Environ Sci Technol, 2015, 49(3):1713-1720
21王硕,于水利,徐巧,付强,李激.好氧颗粒污泥特性、应用及形成机理研究进展[J].应用与环境生物学报,2014, 20(4):732-742[Wang S, Yu SL, Xu Q, Fu Q, Li J. Characteristics, application and formation mechanisms of aerobic granular sludge:recent advances[J]. Chin J Appl Environ Biol, 2014, 20(4):732-742]
22 More TT, Yadav JSS, Yan S, Tyagi RD, Surampalli RY. Extracellular polymeric substances of bacteria and their potential environmental applications[J]. J Environ Manage, 2014, 144:1-25
23 Bafana A. Characterization and optimization of production of exopolysaccharide from Chlamydomonas reinhardtii[J]. Carbohydr.Polym, 2013, 95(2):746-752
24 Fernandez M, Sanchez J. Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451[J]. J Microbiol Meth, 2002, 49(2):207-208
25 Dai YF, Xiao Y, Zhang EH, Liu LD, Qiu L, You LX, DummiMahadevan G, Chen BL, Zhao F. Effective methods for extracting extracellular polymeric substances from Shewanella oneidensis MR-1[J]. Water Sci Technol, 2016, 74(12):2987-2996
26 Kang F, Qu X, Alvarez PJ, Zhu D. Extracellular saccharide-mediated reduction of Au3+to gold nanoparticles:new insights for heavy metals biomineralization on microbial surfaces[J]. Environ Sci Technol, 2017,51(5):2776-2785
27张宇,梁彦秋,贾春云,姜春阳,焦阳.不同方法提取PAHs高效降解菌EPS的特性[J].生态学杂志,2014, 33(4):1027-1033[Zhang Y, Lang YQ, Jia CY, Jiang CY, Jiao Y. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods[J]. Chin JEcol, 2014, 33(4):1027-1033]
28 Hou J, Yang Y, Wang P, Wang C, Lu M, Wang X, Lu B, You G, Liu Z.Effects of CeO2, CuO, and ZnO nanoparticles on physiological features of Microcystis aeruginosa and the production and composition of extracellular polymeric substances[J]. Environ Sci Pollut Res Int, 2017,24(1):226-235
29 Xu H, Jiang H, Yu G, Yang L. Towards understanding the role of extracellular polymeric substances in cyanobacterial Microcystis aggregation and mucilaginous bloom formation[J]. Chemosphere, 2014,117:815-822
30杨敏.微生物胞外聚合物的生成及其对树脂特性的影响研究[D].昆明:昆明理工大学,2012[Yang M. The generation of microbial extracellular polymeric substances and its influence on resin characteristic[D].Kunming:Kunming University of Science of Technology, 2012]
31 Liu L,Qin B,Zhang Y, Zhu G,Gao G, Huang Q, Yao X. Extraction and characterization of bound extracellular polymeric substances fromcultured pure cyanobacterium(Microcystis wesenbergii)[J]. J Environ Sci, 2014, 26(8):1725-1732
32 Wang LL,Wang LF,Ren XM,Ye XD,Li WW,Yuan SJ,Sun M,Sheng GP, Yu HQ, Wang XK. pH dependence of structure and surface properties of microbial EPS[J]. Environ Sci Technol, 2012, 46(2):737-744
33 Morgan JW, Forster CF, Evison L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges[J]. Water Res, 1990, 24(6):743-750
34 Sun M, Li WW, Mu ZX, Wang HL, Yu HQ, Li YY, Harada H. Selection of effective methods for extracting extracellular polymeric substances(EPSs)from Bacillus megaterium TF10[J]. Sep Purif Technol, 2012, 95:216-221
35郑昊.莱茵衣藻对纳米氧化铜在水环境中的分布规律及去除效能的影响[D].泰安:山东农业大学,2016[Zheng H. Effects of Chlamydomonas reinhardtii on removal and distribution of CuO nanoparticle[D]. Tai'an:Shandong Agricultural University, 2016]
36 Zhou K, Hu Y, Zhang L, Yang K, Lin D. The role of exopolymeric substances in the bioaccumulation and toxicity of Ag nanoparticles to algae[J]. Sci Rep, 2016, 6:32998-33009
37慕亚南.自然水体生物膜及其胞外聚合物对Cu2+的吸附研究[D].无锡:江南大学,2016[Mu YN. Adsorption of Cu2+by natural biofilms associated with extracellular polymeric substances(EPS)[D]. Wuxi:Jiangnan University,2016]
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